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My book "Master Control Genes in Developmentand Evolution: The Homeobox Story",
that was published in 1998, summarizes our work on homeotic genes
over the last thirty years. There are still many open questions, but in
the past two years, my research group has solved a few more pieces of the puzzle.
We have pursued the question of how the two homeotic genes Antennapedia (Antp)
and Sex combs reduced (Scr) which have very similar homeoboxes are differentially
regulated. Earlier work had narrowed down the differences between the two proteins
to four amino acid residues in the flexible N-terminal arm of the homeodomain.
Two of these amino acids are threonine 6, that is conserved from flies to humans,
and serine 7, which in vertebrates is replaced by alanine. The regulatory subunit of
the serine-threonine protein phosphatase PP2a of Drosophila binds to the homeodomain
of SCR, but not to thatof ANTP, raising the possibility that SCR is regulated by
a phosphorylation - dephosphorylation mechanism. The mutant protein in which
threonine and serine are replaced by two alanines remains active in transgenic
flies, where as the substitution by two aspartate residues (ressembling the
phosphorylated form) inactivates SCR. This implies that the dephosphorylated
form of the protein is active. RNA interference experiments by injection of
double stranded RNA of the protein phosphatase subunit gives an Scr mutant phenotype,
thus confirming the in vivo regulation of Scr by the protein phosphatase.
The Notch signaling pathway defines an evolutionary conserved cell-cell
interaction mechanism which throughout development controls the ability of precursor cells to
respond to developmental signals. Notch is also involvedin a common regulatory
pathway for the determination of the various Drosophila appendages,
if activated at anearly time point in the developmental pathway. By ectopic
expression of a constitutively activated form of the Notch receptor, Notch
signaling initiates the expression of the master control genes eyeless, vestigial,
Distal-less and homothorax, which in combination with homeotic genes induce the
formation of ectopic eyes, wings, legs and antennae. The regulatory mechanism
for these spectacular effects is under investigation.
In an attempt to reconstruct the evolutionary history of
the homeobox genes, we have isolated and characterized
the Hox genes of the ribbonworm (Lineus sanguineus).The bodyplan of the
ribbonworms (nemertini) seems to be close to the common ancestral condition
found in protostomes and deuterostomes. We found that the Lineus genome contains
a compact Hox cluster of at least 6 genes. Each of these genes can be definitely
assigned to an ortholog group on the basis of its homeobox and its
flanking sequences. The most closely related homeodomain sequences
are invariably found among the mouse and Amphioxus orthologs, rather than
Drosophila and other invertebrates. This suggests that the ribbonworms
may have diverged relatively little from the last common
ancestors of proto- and deuterostomes, the urbilateria.
Lineus also has a remarkable potential for regeneration.
We have artificially generated "homeotic" ribbonworms with a
duplicated ocellar region, replacing the postocellar region
anterior to the brain. Such chimaeras are capable of morphogenetic
regulation to restore the normal antero-posterior pattern.
The missing postocellar region is restored by intercalary regeneration and the
anterior duplicated ocellar region is eliminated by a process
called transgeneration. This reverse homeosis involves
the removal of the syngeneic eyes and the retention of
the grafted allogeneic eyes. The mechanism of this reverse homeosis
remains to be elucidated, but homeobox genes are likely to be involved.
Morphogenesis and evolution of the eyes
Our work on Pax6 as a master control gene for eyemorphogenesis and
the implications for eye evolution
have been summarized for Trends in Genetics (Gehring
& Ikeo, 1999). Pax genes from various animal phyla are
capable of inducing ectopic eye morphogenesis, indicating
that Pax6 is a master control gene for eye development.
It is proposed that contrary to the dogma, the various eye-types
found in metazoa, are derived monophyletically, from a common
prototype. In order to explain the
evolution of the different types of eyes, we have advanced the
hypothesis that different genes are intercalated into the
prototypic eye developmental pathway in the various animal phyla.
In Drosophila we have documented two cases of gene intercalation
(recruitment) into the eye developmental
pathway: twin of eyeless and drosocrystallin. The Drosophila
genome contains in addition to eyeless (ey) asecond Pax6 homolog,
called twin of eyeless (toy). This
pair of genes has presumably arisen by gene duplication
relatively late during insect evolution. toy and ey share a similar
pattern of expression, but toy is expressed much earlier during embryogenesis.
Targeted expression of toy, like ey, induces the formation of ectopic eyes.
Genetic and biochemical evidence indicates, however, that toy functions
upstream of ey by directly regulating the eye-specific enhancer of ey.
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Toy is therefore required forinitiation of ey expression in the
embryo and acts through ey to induce the eye developmental program.
A second gene which appears to have been recruited
into the eye developmental pathway is the major lens
protein gene encoding drosocrystallin. The cloning and
sequencing of this gene clearly indicates that this gene
originally encoded a cuticle protein, that subsequently
was used as a lens protein.
To infer the evolutionary history of Pax6 we have also cloned
the orthologs from Amphioxus and from the planarian Dugesia tigrina.
The Amphioxus Pax6 gene is expressed in the olfactory epithelium,
the lamellar body (a presumed homolog of the vertebrate pineal eye) and the
precursor cells of the frontal eye. The Dugesia Pax6 gene is expressed in
the eyes, both in the photoreceptors and the pigment cells, strengthening
the correlation between Pax6 and eye morphogenesis.
Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W.J., and
Walldorf, U.: Isolation of a Drosophila homolog of the vertebrate
homeobox gene Rx and its possible role in brain
and eye development. Proc. Natl. Acad. Sci. USA 95,
2343-2348 (1998).
Kmita-Cunisse, M., Loosli, F., Bi rne, J., and Gehring, W.J.:
Homeobox genes in the ribbonworm Lineus sanguineus:
Evolutionary implications. Proc. Natl. Acad.Sci. USA 95,
3030-3035 (1998).
Girard, F., Bello, B., Laemmli, U.K., and Gehring, W.J.: In
vivo analysis of Scaffold-associated regions in Drosophila:
a synthetic high-affinity SAR binding protein suppresses
position effect variegation. EMBO J. 17, 2079-2085 (1998).
Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U.,
and Gehring, W.J.: Eyeless initiates the expression of
both sine oculis and eyes absent during Drosophila compound
eye development. Development 125, 2181-2191
(1998).
Bello, B., Resendez-Perez, D., and Gehring, W.J.: Spatial and
temporal targeting of gene expression in Drosophila by
means of a tetracycline-dependent transactivator system.
Development 125, 2193-2202 (1998).
Glardon, S., Holland, L.Z., Gehring, W.J., and Holland. N.D.:
Isolation and developmental expression of the amphioxus
Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor
evolution. Development 125, 2701-2710 (1998).
Leuzinger, S. Hirth, F., Gerlich, D., Acampora, D., Simeone,
A., Gehring, W.J., Finkelstein, R., Furukubo-Tokunaga, K.,
and Reichert, H.: Equivalence of the fly orthodenticle gene
and the human OTX genes in embryonic brain development of
Drosophila. Development 125, 1703-1710 (1998).
Gehring, W.J.: Master Control Genes in Development and
Evolution: The Homeobox Story. Yale University Press
New Haven, USA, 1998.
Callerts, P., Munoz-Marmol, A.M., Glardon, S., Castillo, E.,
Sun, H., Li, W.-H., Gehring, W.J., and Salo, E.: Isolation
and expression of a Pax-6 gene in the regenerating and
intact Planarian Dugesia(G)tigrina. Proc. Natl. Acad. Sci.
USA 96, 558-563 (1999).
Hauck, B., Gehring, W.J., and Walldorf, U.: Functional analysis
of an eye specific enhancer of the eyeless gene in
Drosophila. Proc. Natl. Acad. Sci USA 96, 564-569 (1999).
Janssens, H., and Gehring, W.J.: Isolation and Characterization
of drosocrystallin, a Lens Crystallin Gene of Drosophila melanogaster.
Dev. Biol. 207, 204-214 (1999).
Czerny, T., Halder, G., Callaerts, P., Kloter, U., Souabni, A.,
Gehring, W.J., and Busslinger, M.: Twin of eyeless, a second Pax-6 gene
of Drosophila, acts upstream of eyeless in the control of eye development.
Mol. Cell 3, 297-307 (1999).
Niimi, T., Seimiya, M., Kloter, U., Flister, S. and Gehring,
W.J.: Direct regulatory interaction of the eyeless protein
with an eye-specific enhancer in the sine oculis gene
during eye induction in Drosophila. Development 126,
2253-2260 (1999).
Tarpin, M., Gehring W.J., and Biérne J.: Reverse homeosis
in homeotically reconstructed ribbonworms. Proc. Natl.
Acad. Sci. USA, 96 11900-11903 (1999).
Kurata, S., Go, M.J., Artavanis-Tsakonas, S., and Gehring,
W.J.: Notch signalling and the determination of appendage identity.
Proc. Natl. Acad. Sci. USA, in press.
Gehring, W.J., and Ikeo, K.: Pax 6. Mastering eye morphogenesis
and eye evolution. Trends in Genetics 15, 371-377 (1999).
Gröger H., Callaerts P., Gehring, W.J., Schmid, V.: Gene
Duplication and Recruitment of a Specific Tropomyosin into
Striated Muscle Cells in the Jellyfish Podocoryne carnea.
J. Exp. Zool. (Mol Dev Evol) 285, 378-386 (1999).
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